Phytochemurry, Vol. 30, No. 9. pp. 3021 302s. 1991 Primed in Great Britain.

A C13-NORISOPRENOID

0

GENTIOBIOSIDE

FROM

0031-9422/91 S3.00+0.00 1991 Pcrgamon Press plc

QUINCE

FRUIT

PETERWINTERHALTER, SVEN HARMSENand FRANCOTRANI Lehrstuhlfir Lebensmittelchemie,Universitiit Wiirzburg,Am Hubland 8700 Wtirzburg, Germany (Received

Key Word Index-Cydonia

oblonga;

11 January 1991)

Rosaceae; quince fruit; glycosides; aroma precursors; 3-hydroxy-p-ionol

B-o-gentiobioside; mannelolactones.

Abstract-The

/?-D-gentiobioside [fi-D-ghtcopyranosyl( l-*6)-/_?-D-ghtcopyranoside] of 3-hydroxy-/?-ion01 has been isolated and characterized in quince (Cydonio oblonga) fruit through spectral and chemical studies. Model experiments carried out with this new natural compound revealed its important role as precursor of a number of C, ,-norisoprenoid flavour compounds of quince essential oil.

INTRODUCTION

A considerable number of ionone-related compounds has been reported as constituents of quince essential oil [l, 23. Recent studies by our group [3-S] have shown that these compounds are not original volatiles of quince fruit, but rather are formed during technological processes, e.g. heating, by degradation of acid-labile, nonvolatile precursor compounds. The main C13-norisoprenoids that are formed upon heat-treatment at natural pH conditions of quince juice (pH 3.5) are 2,2,6,7tetramethylbicyclo[4.3.O]nona-4,7,9(l)-triene (l), 3,4didehydro+ionol (t), and 2,2,6,7+etramethylbicyclo[4.3.0]nona-4,9(:1)-dien-8-ol (3), which are present in thermally treated quince juice at a collective concentration of ca 10 ppm. We describe the isolation and structural elucidation of a natural glycosidic precursor of flavour compounds l-3. Furthermore, we report on biomimetic degradation studies carried out with the newly identified glycoside rationalizing the origin of the main C, ,-norisoprenoids of quince essential oil.

RFSL’LTS AND DlSCU!S!3ION

The glycosidic fraction from quince fruit was isolated by passing neutralized quince juice through a column of XAD-2 resin [6]. After elution with methanol the glycosidic fraction was subjected to a prefractionation using rotation locular countercurrent chromatography (RLCC) [7]. The RLCC-system was operated in the ascending mode employing a solvent system that eluted the most polar constituents first from the RLCC appar-

atus. In the collected RLCC-fractions the precursor compounds were indirectly quantified by analysis of their volatile degradation products formed by simultaneous distillation/extraction (SDE)-treatment [8, 91. For these experiments the fractions were pooled into seven groups and after addition of an internal standard an atmospheric pressure SDE at pH 3.5 was carried out on aliquots from each of these pooled groups. The extracts so obtained were then analysed and quantified by HRGC and HRGC-MS. Data for this experiment are shown in Fig. 1, in which the observed relative concentrations of thermally generated flavour compounds in the separated RLCC-fractions are shown. Importantly, besides the generation of norisoprenoids 1-3, formation of the so-called marmelolactones 4a/h was also observed. These isomeric monoterpene Iactones were first isolated from quince (“marmelo” in Japanese) by Japanese researchers [lo, 1l] and were reported to be responsible for the characteristic odour of the fruit. Our results now indicate that these key monoterpene flavour compounds are, like the norisoprenoids 1-3, not original volatiles of intact quince fruits but formed by thermal degradation of non-volatile, presumably glycosidically bound precursor compounds. Good resolution of the progenitors of the Cr3-compounds l-3 and marmelolactones 4a/h is shown in Fig. 1; 86% of the total amount of l-3 was formed from combined RLCC-fractions 5-14, and 91% of the lactones 4a/h was obtained from combined RLCC-fractions 15-24. In an effort to further purify the precursor compound of CIJ-norisoprenoids l-3 combined RLCC-fractions 5-14 were concentrated, acetylated with acetic anhydridepyridine and subjected to flash-chromatography

h/b

3021

3022

P.

-.

_._.

..--=

NORISOPRENOIOS

l-3-

/c-.

-.----

WINTERHALTER

_~

MARMELOLACTONES

4

46 --.

.--

’

60 70

I

50

.:

40

30

-’

20

j

0”

,

1

5-9

l-4

SDE

lo-14

(pli

3.5)

16-19

20-24

treated

25-29

30-34

RLCC-fractions

Fig. 1. Distribution of precursors of C,,-norisoprenoid volatiles l-3 and marmelolactones 4a/b in SDE-treated (pH 3.5) fractions, separated by RLCC, of a glycosidic quince fruit extract. on silica gel. A final purification was achieved by preparative HPLC on silica gel, which yielded an acetylated precursor as a powder. The characterization of the isolated glycoside was made by mass spectrometry as well as ‘H and 13C NMR

et al.

spectroscopy. In the EI mass spectrum prominent ions were obtained at m/z 43, 109, 169, and 331 which are characteristic of acetylated hexoses. Furthermore, a fragment m/z 619 indicated that the sugar moiety is a disaccharide. Because no molecular ion peak was observed in the El mass spectrum the sample was subjected to thermospray-mass spectral analysis, which yielded a fairly strong pseudo-molecular ion at m/i 888 [M + NH4]+. This indicated a molecular mass of 870. From both ‘H (Table 1) as well as 13C NMR data (Table 2), the presence of a disaccharide moiety was confirmed. The ‘H NMR spectrum exhibited two doublets at 64.58 (J= 7.8 Hz) and 4.61 (J = 7.8 Hz) for two anomeric protons, indicating the presence of two fi-glycosidic linkages. Furthermore, from the ‘% NMR data obtained for the unknown sugar moiety a coincidence with data published for perO-acetylated /I-gentiobiose [12] was evident. Therefore, authentic &gentiobiose as well as amygdalin, the /I-gentiobioside of mandelonitrile, were acetylated. The “CNMR spectral data for these two reference compounds, which are also included in Table 2, were in good agreement with the data obtained for the unknown sugar moiety. For the identification of the aglycone the isolated glycoside was deacetylated and enzymatically hydrolysed using /?-glucosidase from sweet almond (emulsin). This treatment liberated the C, ,-norisoprenoid diol, 3-hydroxy+ionol (6b), whose structure was confirmed by synthesis according to the method of Isoe et al. [ 133. The residue of the enzymatic hydrolysis was lyophilized and subsequently permethylated [ 143. Comparative HRGC and HRGC-MS analyses of the permethylated residue with several permethylated reference compounds revealed that only /?-D-glucopyranose, and no fi-gen-

Table 1. ‘H NMR spectral data of the isolated glycoside Sa and of reference compounds 200 MHz, C.DCI,)

_.

Isolated glycoside 5a --

6a .-...

b (ppm)

Signal

Assignment

6 (ppm)

0.99, 1.00 I .33 1.4-1.5 1.64 1.81 I .98-2.08 2.29 3.s3.84 4.10 4.24 4.58’ 4.61’ 4.84-5.23

6H, 2s 3H, d (6.1) I H, dd’ 3H, .s IH, m 25H, m 1H, dd (17.0, 5.4) 4H, m IH, dd (12.3, 2.3) lH, dd; (12.4, 4.8) 1H, d (7.8) 1H, d (7.8) 6H. m

0.99, 1.00 1.33 1.42 I .65 1.73

5.34 5.38 6.04

1H, dq (6.7, 6.1) IH, dd (13.5, 6.7) 1H. br d (13.5)

2Me-1 Me-9 H,-2 Me-5 H,-2 8 x COMe and H,-4 H,-4 H,-G6, H,-G6, H-G5, H-G5’ H.-G6 H,-G6 H-G1 H-GI’ H-G2, H-G2’, H-G3, H-G3’, H-G4, H-G4 H-9 H-8 H-7

Coupling constants (J in Hz) in parentheses. Assignments were made with the aid of authentic reference compounds: B peracetylated /3-u-gentiobiosyl part of amygdalin. “Interchangeable values. *Coupling constants were obscure, due to partial overlap.

(6 relative to TMS,

B ..-.

.-.

.-. 6 (ppm)

1.95-2.08 2.32 3.s3.86 4.10 4.26 4.40 4.65 4.83-5.30 5.35 5.39 6.04

60 3-hydroxy+ionylacetate,

and

C,,-Norisoprenoid

glycoside from quince

3023

Table 2. ‘% NMR spectral data (50 MHz, CDC13) of the isolated glycoside 51 and reference compounds 6a

A ~~

B

DEPT

6 (ppm)

6 (ppm)

6 (ppm)

C

36.71 48.12 64.93 42.07 126.11 136.35 129.12 133.66 71.44 21.08 29.91 28.31 21.33

Isolated glycoside !ta Position

1

a* (ppm)

12’ 13

36.41 45.31 12.70 38.43 124.89 137.02 128.86 133.83 71.49 21.15 29.71 28.44 21.33

G-l G-2b G-3 G-4’ G-5 G-6 G-l’ G-2’b G-3’ G-4” G-5’ G-6

98.60 71.11 12.17 69.13 13.42 68.04 100.59 71.28 12.30 68.31 11.93 61.83

2 3 4 5 6 7 8 9 10

1I’

CO-Me CO-Me

20.57 169.15 - 170.57

CHI CH CHz C C CH CH CH CH3 CH3 CH3 CH3

CH CH CH CH CH CH, CH CH CH CH CH CH, CH, C

20.5 170.34

91.53 10.20 72.78 68.36 13.84 67.42 100.55 70.84 72.61 68.28 71.84 61.78

97.94 70.90 72.51 68.79 13.72 67.78 100.44 71.37 12.42 68.23 71.90 61.76

20.5 168.12 - 170.54

20.5 168.88 - 110.45

*Chemical shifts were assigned on the basis of a DEPT experiment and of comparison with reference substances: 6a 3-hydroxy+ionylacetate, A /?-Dgentiobiose octaacetate and B peracetylated j?-n-gentiobiosyl moiety of amygdalin. ‘-‘Interchangeable values.

tiobiose, was liberated by treatment with emulsin. This can be explained by the fact that emulsin cleaves /?-linked glucose units, as has been shown for amygdalin [15], in a stepwise manner from the non-reducing end of the sugar chain. In a similar manner, if the isolated glycoside was a /?-gentiobioside, it would be hydrolysed in two steps liberating two molecules /‘7-D-ghtcose.On the other hand mild acid hydrolysis would give less complete cleavage products and generate /?-gentiobiose. Therefore, a partial hydrolysis of the deacetylated glycoside Sb under mild conditions according to the method of Shipchandler and Soine [16] was carried out using a cationexchange resin in the acid cycle. The hydrolysate soobtained was lyophilized, permethylated and examined by HRGC and HRGC-MS. These analyses revealed a main hydrolytic product possessing the same retention time and the same mass spectrum as obtained for authentic permethylated /I-D-gentiobiose. Finally, the location of the gentiobiose residue on the aglycone was confirmed as follows. In the “CNMR spectrum the C-3 carbon of the acetylated glycoconjugated diol 6 resonated downfield (by+78 ppm) from

the corresponding signal for free diol. In contrast, the signals for C-2 and C-4 of the acetylated glycoside of 6 resonated upfield by - 2.8 and - 3.7 ppm, respectively. From these data the structure of the isolated glycoside could be assigned as being the /?-D-gentiobioside of 3hydroxy-/3-ionol with the sugar attached to the hydroxyl group in the C-3 position (cf. structure). This glycoside is reported as a natural product for the first time. Moreover, it is the first report of a conjugated form of 3-hydroxy-/_Iionol (6b). a carotenoid-derived component which, in addition to quince, was previously only known as a free aroma component of tobacco [17]. The sugar B-Dgentiobiose [/?-D-ghtcopyranosyt)(1 -r6)-/?-D-gfucopyranoside], however, is a rather common plant constituent, e.g. it is present in the well-known amygdalin, a cyanogenie glycoside occurring in the kernels and seeds of members of the Rosaceae family: almond, apple, apricot, cherry, peach, pear, plum and quince [18]. In a further experiment the deacetylated glycoside 91 was subjected to an atmospheric SDE at natural pH conditions (pH 3.5) of quince juice. The result of this treatment compared to the volatile composition obtained

P. WINTERHALTER rr (11.

3024

for quince juice when treated under the same experimental conditions are shown in Fig. 2. It is obvious that the degradation of the isolated glycoside yielded essentially the same pattern of CIJ-norisoprenoid volatiles as was found in heated quince fruit juice [3]. Besides the main degradation products 1-3, unidentified C,,-hydrocarbons (M, 174) were also observed. Such compounds have been recently found in tomato paste Cl93 and starfruit [20]. Their structural elucidation, together with studies regarding the nature of the conjugates giving rise to the formation of lactones 4a/b, will remain the subject for continuing research. EXPERIMENTAL General.

‘H:

200.13 MHz,

“C: 50.32 MHz For flash chromatography [Zl] Merck silica gel 60 (0.0324.063 mm) was used. All solvents were of high purity at purchase and were redistilled before use. Capillary gas chromatography (HRGC): WCOT fused silica capillary columns: (a) J&W DB-Wax (30 m x 0.25 mm i.d.. film thickness 0.25 pm): (b) J&W DB-1701 (30 m x 0.32 mm i.d., film thickness 0.25 pm). Split injection (1: 20) was used. The temp. programs were 3 min isothermal at 50. and then Increased with 4’ min- ’ up lo 240’ (a) and 1 min isothermal a( 60” and then Increased with 4- mn-’ up to 270’ (b). The flow rates for the carrier gas were 2.0 ml min _ ’ of He, for the make-up gas 30 ml min 1 N,, and for the detector gases 30 ml min. ’ H, and 300 ml min- ’ air, respectively. The injector temp. was kept at 220’ and the detector temp. at 250” (a) and 280- (b), respectively. Capillary GC-MS. The same type of columns as mentioned above for GC analysis were used. The conditions were as follows: temp. programs, (a) from 40’ 10 230 with 4‘ min _ I: (b) from 50- lo 270’ with 3.5 min-‘. Carrier gas flow rate 2.5 ml min _ ’ of He; temp. of ion source and all conncction parts. 2m; electron energy. 70eV; cathodic current, 0.7 mA. Thermospray mass spectrometry: thermospray-bypass interface jet 220 (0.05 M NH,Ac). Positive ions over a range of rn,‘-_ 123 to 900 were scanned. f.so/afion of qlpw.sides. After removing seeds from 10 kg of fresh ripe quince fruit (C. oblonga, Mill.) and cutting into small pieces. fruits were submerged in 5 I 0.2 M PI buffer (pH 7.0, containing 0.2 M glucono-6-lactone [22] as glycosidase inhibitor). homogenized with a blender and pressed. The clear juice obtained was passed through a column (50 cm x 5 cm i.d.) of Ambcrlite XAD-2 resin [6]. After rinsing the column with 21.

SS R = AC Sb R = H PR

6a 6b

R = AC R = H

H,O the glycosidic fr. was eluted with 2 1 MeOH. Careful concn of the MeOH cluate in CLICIAO, followed by Et,0 extraction to ensure removal of any volatlles, gave cu 3 g of a dark brown residue, which was subjected lo a prefractionation using RLCC. The RLCC apparatus (Eyela RLCC UP-60, Tokyo Rikakikai Co.) was operated in the ascending mode employing a solvent system made up from the two phases produced by mixing CHCI,-MeOH-H*O (7: 13:8) with the more dense, less polar layer used as the stationary phase. The Row rate was 1 ml min _ ‘, rotation speed was 80 rpm, slope 30’. 34 frs (each 10 ml) were collected. For the work-up of the glycosidic isolate two separations (each 1.5 g) were carried out. Scrrenmg r)jHLCC-fracrions. For monitoring compounds l-4 in sepd RLCC-frs closely spaced frs were pooled in groups of five. Part (1iSO th) of each pooled fr. was &solved in 0. I M citric acid/Pi buffer (pH 3.5. 30 ml) and this soln, after addition of 2-heptanol (50 pg) as int. standard was subjected to atm. pres. simultaneous distillatlon;extractlon (SDE) with pentane--E120 (I : 1) as solvent. SDE treatment was performed over I hr using the SDE head described by Schultz er ul. [S]. The organic layer was dried over NazSOI and carefully coned. 10 50~1 prior lo GC and GC-MS analysis. Dericarirufion and purification of prracerylared glycosides. Combined RLCC-frs 5-14 were acetylated with Ac,C-pyridine at room temp. The acetates obtained were further separated by flash chromatography on silica gel (pentane- Et,0 gradient). Final purification by scmiprep. HPLC (LiChrosphere Si100.

9

/I

II,1

a

J

Fig. 2 GC-separation (J&W WCOT fused silica column DBWax, 30 m x 0.25 mm i.d., film thickness 0.25 pm) of (a) volatrles obtained by thermal degradation (SDE, 2 hr. pH 3.5) of glycoside 5b and (b) quince fruit volatiles Isolated by the SDE method under identical cxperlmental conditions [3]

C , ,-Norisoprenoid 5 pm, i.d. 16 mm; pentane-Et,0 (I :9), flow rate 7 ml min- ‘) of flash fr. 3 gave compound 5s (120 mg) as a powder. ‘H NMR: see Table 1. 13C NMR: see Table 2. Thermospray-MS m/z: 888 [M +NH,]+. EIMS probe(70 eV)m/z(rel. int.): 619 (2). 331(31), 234 (12), 175 (26). 169 (61). 159 (17). 121 (12). 119 (It), 109 (31). and 43 (100). Deocetylation of disaccharide (5a). To 30 mg of Sa in 5 ml MeOH 5 ml 0.02 M Na methanolate soln was added. After 12 hr the mixt. was neutralized by adding 90 mg Dowex 50 WX8 (H+form). After removal of the ion-exchange resin by filtration, and rinsing with MeOH, the solvent was evapd in uacuo and the deacetylated !% was taken up in 3 ml dist. HzO. Enzymatic hydrolysis. To l/3 part (1 ml) of the soln of 5b in 10 ml of 0.2 M citric acid/Pi buffer (pH 5.0) 20 mg almond glucosidase (emulsin) was added. Incubation under Nz was carried out over 24 hr at 37”. The Liberated aglycone 6b was liquid-liquid extracted with Et,0 and subjected to GC and GC-MS analysis. The water layer was lyophilized and the residue was permethylated by the method of ref. [14]. GC and CC-MS analyses showed the presence of a single carbohydrate, possessing the same R, and the same mass spectrum as obtained for an authentic permethylated @-glucopyranose reference. Partial hydrolysis of compound 5b. To a further aliquot (1 ml) 2 ml Hz0 and 50 mg cation-exchange resin (Dowex 5OWX, H+form) were added. The sample was stirred under N, at 80” for 1 hr. After filtration, the water phase was lyophilized, permethylated as described above and subjected to GC and GCMS analysis. These analyses revealed a main product possessing the same R, and identical MS data as found for authentic per methylated fi-D-gentiobiose. Degradation of compound Sb at natural pH-conditions ofquince hit. The remaining aliquot (I ml) of Sb was diluted with 50 ml of 0.2 M citric acid/Pi buffer (pH 3.5) and subjected to SDEtreatment for 2 hr. The organic layer was dried over anhydr. NazSO, and carefully coned. to 50 ~1 prior to GC and GC-MS analysis. Reference compounds. 3-Hydroxy+ionyl acetate (6a) was prepd by the method of ref. [13] and showed the following chromatographic and spectral data: GC: R, (DB-Wax)= 2532; R, (DB-1701)= 1950. UV: 232 nm. EIMS (70 eV) m/z (rel. int.): 234 [M-H,O]+ (1). 192 (9). 177 (11). 159 (41). 144 (9). 133 (22). 119 (19), 105 (24), 91 (28), 77 (16). 55 (16). 43 (100). ‘HNMR (200 MHz, CDC13): 60.99 and 1.00 (6H, 2s. 2Me-l), 1.33 (3H, d, 5=6.2Hz,Me-9), 1.42(1H,dd,5=12.1, 12.0HrH,-2). 1.65(3H, s, Me-5), 1.73(1H,ddd.J= 12.0,3.7,2.0 Hz; H,-2), 1.8-2.0(2H,m, HO-3 and H,-4), 2.02 (3H, s, COMe), 2.32 (IH, dd, J= 17.0, 5.4Hz; H,-4), 3.95 (IH, m, H-3), 5.35 (IH, dq. J=6.9, 6.2 Hz; H-9). 5.39 (lH, dd, J=14.9, 6.9 Ht H-8), 6.04 (lH, br d, J= 14.9 HZ H-7). “CNMR: see Table 2. Deacetylation of compound C as described above gave 3hydroxy-/?-ionol (6b), whose spectral data have been provided previously [S]. For GC and GC-MS studies the following carbohydrate references, i.e. @-glucose, B-D-gentiobiose, and amygdalin, have been derivatti through (a) peracetylation with AczO-pyridine at room temp., and (b) permethylation by

3025

glycoside from quince the method of ref. [14]. Compounds

l-3 were available from previous studies [3,5], and lactones 4a/b were donated samples. Acknowledgements-We thank Mrs Colberg (Institut fir Pharmakologie und Toxikologie, Universitiit Wiirzburg) for recording the thermospray-MS spectrum and Dr Takeoka (USDA, ARS, Albany, California) for generously providing a sample of marmelolactones. REFERENCES 1. Tsuneya, T., Ishihara, M., Shiota, H. and Shiga, M. (1983) Agric. Biol. Chem. 41, 2495.

2. Ishihara, M., Tsuneya, T., Shiota, H., Shiga, M. and Nakatsu, K. (1986) J. Org. Chem. 51, 491. 3. Winterhalter, P., Lander, V. and Schreier, P. (1987) J. Agric. Food Chem. 35, 335. 4. Winterhalter, P. and Schreier, P. (1988) J. Agric. Food Chem. 36,560.

5. Winterhalter, P. and Schreier, P. (1988) J. Agric. Food Chem. 36, 1251. 6. Giinata, Y. Z., Bayonove. C. L., Baumes, R. L. and Cordonnier, R. E. (1985) J. Chromatogr. 331, 83. 7. Snyder, J. K., Nakanishi, K., Hostettmann; K. and Hostettmann, M. (1984) J. Liq. Chrotnatogr. 7, 243. 8. SchultG T. H., Flath, R. A., Mon, T. R., Eggling. S. B. and Teranishi, R. (1977) J. Agric. Food Chem. 25, 446. 9. Winterhalter, P., Sefton, M. A. and Williams, P. J. (1990) Am. J. Enol. Vitic. (in press). 10. Tsuneya, T., Ishihara, M.. Shiota, H. and Shiga, M. (1980) Agric. Biol. Chem. 44, 957. 11. Ishihara, M., Tsuneya, T., Shiota, H., Shiga, M. and Yokoyama, Y. (1983) Agric. Biol. Chem. 47, 2121. 12. Bock, K., Pedersen, C. and Pedersen, H. (1984) Adu. Carbohydr. Chem. Biochem. 42, 193. 13. Isoe, S., Katsumura, S., Be Hyeon. S. and Sakan, T. (1971) Tetrahedron Letters 1089. 14. Finne, J., Krusius, T. and Rauvala, H. (1980) Curbohydr. Res. 80, 336. 15. Haisman, D. R. and Knight, D. J. (1967) B&hem. J. 103, 528. 16. Shipchandler, M. and Soine, T. 0. (1968) J. Pharm. Sci. 57, 747. 17. Fujimori, T., Kasuga, R., Kaneko, H. and Noguchi, M. (1975) Agric. Biol. Chem. 39, 913.

18. Cairns, T., Frober& J. E., Gonzales, S., Langham, W. S.. Stamp, J. J., Howle, J. K. and Sawyer, D. T. (1978) Analal. Gem. so, 317. 19. Buttery, R. G., Teranishi, R., Flath, R. A. and Ling, L. C. (1990) J. Agric. Food Chem. 38, 792. 20. MacLeod, G. and Ame=s,J. M. (1990) Phyrochemistry 29, 165. 21. Still, W. C., Kahn, M. and Mitra, A. (1978) J. Org. Chem. 43, 2923. 22. Hevworth.~,R. ~~ ~~~, ~~and Walker. P. G. (19621 \ I Biochem. J. 83. 331.